Project Summary Increased obesity, and associated metabolic disease, has become one of the most significant public health issues in the modern era. Chronic adipose inflammation links obesity to its metabolic complications. Recent studies indicated that adipose T cells partner with adipose tissue macrophages to cause inflammation and insulin resistance. Targeting the inflammatory T cells, Th1 and CD8+ T cells, by genetic approaches or depletion of T cell populations with antibodies, significantly improves obesity-induced insulin resistance. Limited understanding of the mechanism(s) in obesity-induced adipose T cell activation impedes the development of an effective and specific means to uncouple obesity from its negative effects on human health. T cell activation requires major histocompatibility complex (MHC)-mediated antigen presentation and co- stimulation. Whereas MHC participates in cell activation in almost all T cell-mediated immune responses, individual members of the large family of co-stimulators plays specific roles in certain immune responses. This diversity of co-stimulators makes them attractive therapeutic targets in immune-related diseases. Adipocyte MHC class II (MHCII) plays an essential role in obesity-induced CD4+ adipose T cell activation and insulin resistance, and adipose tissue macrophage (ATM) MHCII may play a similar role. A T cell co-stimulator gene expression profile in adipocytes and ATMs from lean and obese mice revealed that OX40 ligand (OX40L) is the most profoundly upregulated T cell co-stimulator in both cell types during obesity. Importantly, OX40L levels are also increased in adipocytes from obese and insulin-resistant human patients. OX40L only binds to a single receptor, OX40, and OX40-/- mice on a high fat diet demonstrated attenuated Th1 and CD8+ T cell activation in adipose tissue and significant improvements in insulin resistance. Based on these results, the hypothesis that OX40L- OX40 co-stimulation mediates obesity-induced activation of adipose Th1 and CD8+ T cells, promoting insulin resistance, will be tested in this proposal using a variety of cellular and genetic techniques. Adipocyte- and macrophage-specific knockout mice will be used to determine if adipose OX40L is necessary and sufficient for adipose T cell activation and insulin resistance in a diet-induced obesity mouse model. Furthermore, the mechanisms by which adipose Th1 and CD8+ T cells are reduced in obese OX40-/- mice will be investigated and whether this reduction improves insulin sensitivity in OX40-/- mice will be investigated. Finally, whether OX40L- blocking antibody can prevent or ameliorate obesity-induced inflammation and insulin resistance will be tested. Together, these approaches will reveal whether OX40L/OX40 co-stimulation plays a critical role in obesity- induced inflammation and insulin resistance, and whether proven-safe OX40L blocking antibodies can target and treat obesity-mediated complications.